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Hilditch, T. P. (1956), The Chemical Constitution of Natural Fats, 3rd ed., London, Chapman and Hall, p. 379. Hirayama, N. (1961), J . Agr. Chem. SOC.Japan 35, 437, 441. Holman, R. T., and Hanks, D. P. (1955), J . A m . Oil Chemists’ SOC.32, 356. Horrocks, L. A., and Cornwell, D. G. (1962), J . Lipid Res. 3, 165.
Huang, P. T., Holman, R. T., and Potts, W. (1949), J . A m . Oil Chemists’ SOC.26, 405. Rusoff, I. I., Platt, F. R., Klevens, H. B., and Burr, G. 0. (1945), J . A m . Chem. SOC.67, 673. Schlenk, W. Jr., (1962), Festschr. Carl Wurster 60, Geburtstag, 105. (Chem. Abstracts 57, 14930g.). Scholfield, C. R . , and Hicks, M. A. (1957), J . A m . Oil Chemists’ SOC.34, 77. Stahl, E. (1958), Chemiker Ztg. 82, 323.
Differences among Antibodies Formed in Response to the p-Azobenzenephosphonate and p-Azobenzenearsonate Haptens *
v. PETER KREITERt AND DAVIDPRESSMAN From the Department of Biochemistry Research, Roswell Park Memorial Institute, N. Y.State Department of Health, Buffalo Received July 1, 1963 The relative binding constants of various substituted benzenephosphonates and benzenearsonates for the reaction with rabbit antibodies formed in response to the p-azobenzenephosphonate (P,) haptenic group have been measured by hapten inhibition of specific precipitation. These values exhibit a variation with hapten structure that is distinctly different from that shown with antibodies formed in response to the p-azobenzenearsonate (R,) haptenic group. Although anti-R, antiserum contains two distinct antibodies, one formed in response to the doubly charged ion, the other to the singly charged ion, and anti-P, antiserum apparently contains primarily antibody formed in response to the doubly charged ion, the difference between the two antisera in reaction patterns with various haptens appears to be attributable, for the most part, to a difference between antibodies formed in response to the doubly charged ions. The data are interpreted to mean that the combining site of the anti-P, antibody fits more loosely about doubly charged benzenephosphonate than the combining site of anti-R, antibody fits about doubly charged benzenearsonate. Recent studies (Kreiter and Pressman, 1963) have indicated that anti-R, serum contains two populations of antibodies formed in response to the two ionic forms of p-azobenzenearsonate. In contrast, anti-P, serum contains antibody formed only in response to the doubly charged phosphonate ion. Such a difference between the two antisera suggests that their reaction patterns with various haptens should be different. Although a large amount of information is available on the specificity of antiserum formed in response to the para-azobenzenearsonate group (R, system) (Landsteiner, 1945; Pauling and Pressman, 1945; Erlenmeyer and Berger, 1932), very little information is available regarding the specificity of the antiserum formed in response to the structurally similar paruazobenzenephosphonate group (P, system) except that it reacts strongly with benzenearsonate haptens (Kreiter and Pressman, 1963). The structural similarity between the benzenearsonate and the benzenephosphonate groups was found by Erlenmeyer and Berger (1932) to result in extensive cross reaction of these substances with anti-R, antibody. On this basis it would appear that the specificity of anti-P,, antibodies should closely resemble the specificity of anti-R, antibodies. The relative binding constants for the reactions of several substituted benzenephosphonic acids and benzenearsonic acids with anti-P, and anti-R, rabbit yglobulins have been measured by determining their ability to inhibit precipitation of antibody by the
* Supported in part by a grant (E-2342) from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U. S. Public Health Service. t Present address: Palo Alto Medical Research Foundation, Palo Alto, Calif.
homologous hapten coupled to a foreign protein. A distinct difference in reaction patterns was found between the two systems that could not be attributed to the presence of more than one antibody in anti-R, serum, but rather to a difference in specificity between antibody formed in response to the doubly charged p-azobenzenephosphonateion and antibody formed in response to the doubly charged p-azobenzenearsonate ion. EXPERIMENTAL Antigens.-The antigens (R,-bovine y - globulin and P,-bovine y-globulin used for immunizing rabbits were prepared by coupling diazotized p-aminobenzenearsonic acid and p-aminobenzenephosphonic acid to bovine y-globulin (fraction 11) (Kreiter and Pressman, 1963). Test antigens were prepared by coupling the diazotized amines to ovalbumin. The ovalbumin test antigens were purified by acetone extraction (Nisonoff and Pressman, 1958). Antisera.-Rabbits were injected intravenously with 1 ml of 1% R,-bovine y-globulin antigen three times a week for 3 weeks. The animals were bled 1 week after the last injection and weekly thereafter. One ml of antigen was injected immediately after each bleeding. Anti-P, sera were obtained in a similar manner. The antisera from the first 4 months were pooled according to the titer obtained with the ovalbumin test antigens. A single pool of each antiserum was used in all the experiments. The pool of anti-P, antiserum and the pool of anti-R, antiserum used here were the same as those used in our previous study (Kreiter and Pressman, 1963). The anti-P, pool was obtained from the sera of nine rabbits, three of which contributed equally to about two-thirds of the total
Vol. 3, No. 2, Feb., 1964
275
ANTIBODIES TO p-AZOBENZENEPHOSPHONATE AND p-AZOBENZENEARSONATE
TABLE I COMBINATION OF HAPTENS WITH ANTI-P, ANTIBODY
Hapten
KO'(rel)
AFo (rel) (kcal)
U
Amount of Precipitateasb a t Concentration of Hapten X 10j M 10.4
41.7
167
+0.15 -0.03 -0.17 + O . 13
66 71 68 61 68
41 44 38 33 44
20 22 16 12 21
+0.23 -0.11 +o. 19 +o. 21 -0.27
69 73 82C 72 75 58
43 50 64c 60d 51 35
21 25 39c 39d 26 15
58 49 1008
27 28 79e
5 5 30e
Set 1 Benzenephosphonic acid Unsubstituted o-Chlorom-Chlorop-Chloro3,5-Dichloro-
1.00 0.76 1.05 1.37 0.79
3 3 2.5 2 5 3
Set 2 Benzenephosphonic acid Unsubstituted m-Nitrop-Nitrop-Nitro-o-methoxylm- Aminop-Amino-
1.00 0.66 1.23 0.71 0.68 1.63
3 3 3 4 2.5 3 Set 3
Benzenephosphonic acid Unsubstituted p-Nitrop- (p-Hydroxyphenylazo)Benzenearsonic acid Unsubstituted p-Nitro-
1.00 1.32 7.1
2 2.5 1
-0.15 -1.1
0.31 0.69
2 2.5
+o. 20
86 63
47 36
22 10
+0.24 $0.04 -0.27
76 80 72 70f
55 61 57 48
34 40 36 26
-1.38 -1.81
70 79h 58h7
60 55h 37h
27h 15h
$0.65
Set 4 Benzenearsonic acid Unsubstituted o-Nitrom-Nitrop-Nitro-
1.00 0.65 0.93 1.62
3 3 4 3
Set 5 Benzenearsonic acid Unsubstituted p- (p-Hydroxyphenylazo)p- @-Hydroxyphenylazo) benzenephosphonic acid
1.00 12.3 26.4
3 2.5 2.5
350
a Percentage of the amount of precipitate obtained in the absence of hapten. The amounts of precipitate obtained in the absence of hapten for sets 1-5 are 303 pg, 275 fig, 106 pg, 274 rg, and 274 pg, respectively. b Average deviation from the mean values for the amounts of precipitate obtained with duplicates for sets 1-5 are &4 $6,A2 yo, 1 8%, i l %, and &4%, respectively. Concentrations are l / d of those in the heading. d Concentrations are of those in the heading. e Concentrations are of those in the heading. At a concentration of 8.1 X 10-5 M a value of 80 was observed fl At a concentration of 1 X M a value of 24 was obtained. Concentrations are of those in the heading. A t a concentration of 2.5 X 10-s M a value of 71 was observed.
pool. The anti-R, pool was obtained from the sera of eight rabbits, two of which contributed equally to two-thirds of the pool. y-Globulin fractions were prepared from the pooled antisera by three sodium sulfate precipitations a t room temperature (Nisonoff and Pressman, 1958). y-Globulin prepared in this manner showed less than 5 % of other proteins by freeboundary electrophoresis a t pH 8.6 in barbital buffer. Haptens.-Benzenearsonic acid, benzenephosphonic acid, o-nitrobenzenearsonic acid, and m-nitrobenzenearsonic acid, obtained from Distillation Products Industries, were crystallized from ethanol-water mixture before use. p-Aminobenzenearsonic acid, benzoic acid, and sodium benzenesulfonate were used as received from Distillation Products Industries. pNitrobenzenearsonic acid was prepared by reaction of diazotized p-nitroaniline with sodium arsonite Find purified by repeated precipitations from basic solution with hydrochloric acid and finally by crystallization from water. The preparation of p- (p-hydroxyphenylazo)benzenearsonic acid and p - (p-hydroxyphenylazo benzenephosphonic acid has been described previously (Kreiter and Pressman, 1963). m-Nitro-, p-nitro-, p-nitro-o-methoxy-, m-amino, p-amino, o-chloro, and
p-chlorobenzenephosphonic acids were kindly supplied by Dr. G. 0. Doak. m-Chloro-, and 3,Ei-dichlorobenzenephosphonic acids were obtained from Dr. R. K. Ingham. Stock solutions, 0.01 M, of these compounds were prepared by dissolving a weighed sample in sufficient 0.16 M NaOH to give a pH of 8.0-8.1. These solutions were then diluted with sodium borate buffer, pH 8. Stock solutions, 0.0001 M, of the two azo compounds were prepared by dissolving the compounds directly in borate buffer, pH 8. Solutions of the desired concentrations were prepared from the stock solutions by diluting with borate buffer. Buffer.-The borate buffer used was prepared by mixing 165 ml of a solution of 0.16 M sodium hydroxide with 1 liter of a solution of 0.2 M boric acid containing 0.16 M sodium chloride. The pH of this solution was 7.97 t 0.02 a t 25". Reactions of Antibody with Antigen and Haptens.Solutions of the test antigen were prepared by diluting the stock solution with borate buffer. The concentration (optimum) of test antigen required for maximum precipitation of antibody from a 20 mg, ml solution of y-globulin was determined.
276
Biochemistry
V. PETER KREITER AND DAVID PRESSMAN
TABLE I1 COMBINATION OF HAPTENSWITH ANTI-R, ANTIBODY
A F o (rel)
KO'(rel)
Hapten
U
Amount of Precipitateas * at Concentration of Hapten X lo5 M
(kcal)
10.4
41.7
167
-0.10 -0.69
60 56 65
36 26 34c
9 5
+0.33 -0.23 -0.48 -0.05
68 75 64 51 67
45 58 35 21 41
15 26 11 4 17
+ O . 34 -0.31 -0.85
81d 80 85e 8gf
58d 56 60" 66 f
24 22 27" 35 f
Set 1 Benzenephosphonic acid Unsubstituted m-Nitrop-Nitro-
1.00 1.20 3.46
2.5 2 2
8C
Set 2 Benzenephosphonic acid Unsubstituted o-Chlorom-Chlorop-Chloro3,5-Dichloro-
1.00 0.55 1.52 2.40 1.10
2.5 2.5 2.5 2 2.5
Set 3 Benzenearsonic acid Unsubstituted o-Nitrom-Nitrop-Nitro-
2 2 2 2
1.00 0.54 1.74 4.69
Set 4 Benzenearsonic acid Unsubstituted p-Nitrop- @-Hydroxyphenylazo) Benzenephosphonic acid Unsubstituted p- (p-Hydroxyphenylazo)-
1.00 4.68 12.1
2 2.5 2
-0.85 -1.38
77 71O 76h
52 470 46h
20 190 lBh
1.00 12.3
2.5 2.5
0.0 -1.37
74 73t
51 47i
25 201
a Percentage of the amount of precipitate obtained in the absence of hapten. The amounts of precipitate obtained in the absence of haptens for sets 1-5 are 188 pg, 272 pg, 254 pg, and 322 pg, respectively. b Average deviation from the mean values for the amounts of precipitate obtained with duplicates for sets 1-4 are f 3 %, A 4 yo, f 3 yo,and f 2 yo,respectively. Concentrations are l/4 of those in the heading. Concentrations are of those in the heading. e Concentrations are I / , of those in the heading. Concentrations are of those in the heading. 0 Concentrations are of those in the of those in the heading. At a concentration of 10-3 M a value of 7 was observed. heading. Concentrations are Concentrations are l/10 of those in the heading. At a concentration of 10-3 M a value of 11 was observed.
Experiments on the hapten inhibition of precipitation were carried out by adding 0.20 ml of hapten solution, 0.20 ml of test antigen (optimum concentration), and 0.20 ml of y-globulin to a small tube in that order. Duplicate samples with each hapten were run at three or four concentrations. In order to obtain the amount of precipitate in the absence of the hapten, duplicate samples were run using 0.20 ml of borate buffer, p H 8, in place of the hapten solution. Blanks containing borate buffer, 0.40 ml, and 0.20 ml of y-globulin were run a t the same time to correct for small amounts of protein that precipitate from y-globulin solutions standing a t 3-5". The mixtures were allowed to stand a t 37" for 1 hour and then for 5 days at 3-5". At the end of this time the mixtures were centrifuged, and the precipitates were washed twice with borate buffer and twice with 0.16 M sodium chloride. The washed precipitates were dissolved in 0.02 ml of 0.02 N sodium hydroxide. The solutions were transferred to Klett tubes and diluted with 0.8 ml of water. The protein solutions were analyzed by a modified Folin method (Lowry et al., 1951). The intensity of the color after 1-1.5 hours was measured with a Klett colorimeter using a No. 66 filter. A calibration curve was prepared from y-globulin solutions standardized by measuring the optical density of the y-globulin in borate buffer at 280 mp and taking the optical density of 1 mg/ml to be 1.5.
RESULTS Anti-P, Antibody.-The effects of various haptens on the precipitation of anti-P, and anti-R, antibody
by P,-ovalbumin and R,-ovalbumin, respectively, are given in Tables I and 11. The average relative binding constants K'o (rel) (relative to that of the reference haptens, taken as 1.00) and the heterogeneity constant, U , €or the combination of hapten and antibody were calculated as described previously, using the theory of heterogeneity of combining sites of antibody which assumes a Gaussian distribution of combining energies ( P a w et al., 1944). The values of K'o (rel) have, in general, a precision of about *lo% as shown by duplicate measurements and by the error found in fitting calculated curves (from which the K'o (rel) values are computed) to the experimental points (i.e., plots of the percentage of the amount of precipitate obtained in the absence of hapten vs. the log of the moles of hapten added). The relative AF" values calculated for 5' are listed. The charge specificity of anti-P, antibody is similar to that found €or various other antihapten antibodies (see Landsteiner, 1945) in that anti-P, antibody did react with the structurally similar benzenearsonatehaptens but not with the other charged haptens examined. Neither benzoic acid nor benzenesulfonic acid inhibited the precipitation of anti-P, antibody by P,-ovalbumin (Table 111) at 0.0017 M, the highest concentration used. As shown in Table I, ortho and meta substituents on the benzene ring of benzenephosphonate have little effect on the binding energy of the reaction of the hapten with anti-P, antibody. An ortho-chloro group reduced the binding energy by only 0.15 kcal. Even the larger ortho-methoxy group of p-nitro-o-methoxybenzenephosphonic acid reduced the binding by only
Vol. 3, No. 2,Feb., 1964
277
ANTIBODIES TO p-AZOBENZENEPHOSPHONATE AND p-AZOBEEIZENEARSONATE
TABLE I11 EFFECT OF DIFFERENT ANIONS (1.7mM) ON THE PRECIPITATION OF ANTI-P,ANTIBODYBY P,-OVALBUMIN AT pH 8 Precipitate Found in Absence of Anions Anion
( %)
Benzenephosphonate Benzenearsonate Benzoate Benzenesulfonate
23 40
102 100
0.30 kcal as compared with the binding of p-nitrobenzeneophosphonic acid. A single meta-chloro group had no significant effect and even two meta-chloro groups did not appreciably decrease the binding energy. The larger nitro group in the meta position, although showing a greater effect in reducing the binding than the chloro group, still exhibited only a minor energy contribution of 0.23 kcal. The smaller amino group in the meta position reduced the binding to about the same extent as did the nitro group. The para-@hydroxyphenylazo) group increased the binding energy by 1.1 kcal in accord with earlier experiments with comparable systems (Pressman et al., 1944; Pauling and Pressman, 1945). Other para substituents, nitro and chloro, increased the binding energy by less than 0.20 kcal.1 It is interesting that the para-amino group contributed more energy (0.27 kcal) to the binding than did the para-nitro group, in contrast to the usual observation with other antihapten systems that the nitro group, in the position of attachment of the hapten to the protein of the injected antigen, contributes appreciably more energy than the amino group (Pressman et al., 1944, on anti-p-azobenzoate; Pauling and Pressman, 1945; on anti-p-azobenzenearsonate). The effect of substituents on the binding of benzenearsonate by anti-P, antibody is identical to their effect on the binding of benzenephosphonateafter taking into account the degree of ionization shown in Table IV. The anti-P, antibody present in the pool of anti-P, sera used here had previously been found to consist essentially of antibody formed in response to the doubly charged phosphonate group and reacting only with the doubly charged phosphonate and arsonate ions.2 The apparent relative binding constants in Table I were based on the total amount of arsonate present. These values of KO'(rel) were corrected for the degree of ionization in Table V, where the relative binding constants listed have been calculated on the basis of the concentrations of doubly charged ions only. On this basis the doubly charged benzenearsonate combined just as strongly as the doubly charged benzenephosphonate. The m- and p-nitrobenzenearsonates yielded relative binding constants that were almost the same as were found for the corresponding benzenephosphonates. o-Nitrobenzenephosphonate was ' T h e contribution of the para-nitro group was even smaller than shown in Table I and was apparently zero within experimental error when the KO'(rel) had been corrected for partial ionization of the haptens, assuming that only the doubly charged ions were being bound (see later discussion and Table V). 2 These conclusions do not necessarily mean that there was no antibody formed in response t o the singly charged p-azobenzenephosphonte, but rather that such an antibody was not detected by the means used, Le., reaction with singly charged arsonate ion at pH 6. Such a n antibody, however, if present, was probably present in only relatively small amounts.
TABLE IV DEGREES OF IONIZATION OF BENZENEPHOSPHONATES AND BENZENEARSONATES AT DH 8 Per Cent of Doubly Charged Ion at pH 8 Substituent Unsubstituted o-Chlorom-Chlorop-Chloroo-Nitrom-Nitrop-Nitro0-Aminom-Aminop-Aminop- (p-Hydroxyphenylazo) -
Benzenephosphonate"
Benzenearsonateb
90 91 96 95 95 98 98
25
-
36 22 61 61 18 19 11 56
87 75 98
4 Calculated from ionization constants taken from Jaffe et al. (1953). b Calculated from ionization constants taken from Pressman and Brown (1943).
TABLE V COMPARISON OF BINDING OF NITRO-SUBSTITUTED BENZENEARSONATES AND BENZENEPHOSPHONATES BY ANTI-P, ANTIBODY. CORRECTION FOR PARTIAL IONIZATION OF HAPTEN s
KO'(rel) Uncorrected Benzenephosphonic acid Unsubstituted m-Nitrop-NitroBenzenearsonic Acid Unsubstituted o-Nitrom-Nitrop-Nitro-
Correcteda
1.00 0.66 1.23
1.00 0.58 1.09
0.31 0.20b O.2gb 0.69,0.50b
1.15 0.91b 0 . 45b 1.00,0.73b
a Calculated assuming that only the doubly charged ion is being bound. Different set of measurements where benzenephosphonate was not run. The values for the arsonates are all adjusted t o a standard value of 1.00 for benzenephosphonate using the measured ratios of 1.00j0.31 (uncorrected) and 1.00/1.15 (corrected) for benzenephosphonate/benzenearsonate,respectively.
not available for comparison, but the small effect of the ortho-nitro group in benzenearsonate (Table V) is in agreement with the relatively small effects of other ortho substituents on the binding of benzenephosphonate. Anti-R, Antibody.-The relative binding constants obtained for the reaction of anti-R, antiserum with various benzenearsonates (Table 11) that have been studied previously agree well with the earlier data3 that indicate that the combining site of the anti-R, antibody fits fairly closely about benzenearsonate. Of particular significance is the fact that both P, and R, type antibody, nitro, and chloro4 substituents in ortho and para positions affected the binding energy of benzenephosphonate qualitatively in the 3 The values of KO'(rel) previously obtained for benzenephosphonate and for o-nitro-, m-nitro-, p-nitro-, and p-(phydroxyphenylazo)-bemenearsonates were 0.98, 0.60, 1.5, 5.3, and 13, respectively (Pressman and Siegel, 1953; Pressman et al., 1945). 4 The values of KO'(rel) found for 0-, m-,and p-chlorobenzenearsonates and anti-R, antiserum were 0.30, 1.3, and 2.2, respectively (Pauling and Pressman, 1945).
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Biochemistry
V. PETER KREITER A N D DAVID PRESSMAN
same manner as they affected the binding energy of benzenearsonate. However, the effect of substituents on the binding energy of the reaction of benzenearsonate or benzenephosphonate and anti-R, antibody is seen to be quantitatively different from their effect on the binding energy for the reaction of benzenephonphonate or benzenearsonate with anti-P,, antibody. In the R, system there was not only a larger reduction in binding by ortho groups, than in the P, system, but it is especially noticeable that para groups greatly increased the binding energy while, except for the azophenyl substituent, almost no effect by para groups was seen in the P, system (KO' [rel]corrected for ionization). A metu-nitro group increased the binding energy with anti-Rp antibody while the binding energy was decreased in the case of the anti-Pp antibody (see Table V, corrected KO'[rel]).
DISCUSSION Combining Site of Anti-P, Antibody.-The effect of various substituents on the binding energy of the reaction of benzenephosphonate with anti-P, antibody indicates that the anti-P, combining site fits rather loosely about the doubly charged phosphonate hapten. This loose fit is confirmed by lack of substituent effects on the binding energy of benzenearsonate which combines equally well with anti-P, combining sites. In agreement with a previous observation (Kreiter and Pressman, 1963)) the anti-P, combining sites apparently combine only with the doubly charged arsonate ion, since good correlation of the relative binding constants of benzenearsonates and benzenephosphonates is obtained only if the constants are calculated on the basis of the concentration of the doubly charged ion. The extent of cross reaction of benzenearsonate and benzenephosphonate with the combining sites of the anti-P, antibody is very appreciable in spite of the fact that the arsonate group is larger than the phosphonate group.5 The accommodation of the benzenearsonate by a combining site complementary for the smaller benzenephosphonate therefore indicates that the portion of the combining site complementary to the phosphonate group (POs=)is more spacious than appears to be necessary to accommodate the phosphonate group. Combining Site of the Anti-R, Antibody.-Although the pool of anti-R, antiserum used here has been shown previously to contain some antibodies formed in response to the singly charged p-azobenzenearsonate and others to the doubly charged hapten, only the contribution of the antibody directed against the doubly charged hapten is apparent in these experiments. The strong binding of benzenephosphonates by the y-globulin fraction from anti-R, serum must be due, for the most part, to the binding of doubly charged phosphonate ions, since at p H 8 the benzenephosphonates are essentially all in the doubly charged form and constants calculated on the basis of the small amount of singly charged phosphonate ions present are not reasonable because they are about 15-40 times higher for the heterologous haptens than those observed for the homologous hapten. Moreover, in the case of the homologous hapten itself, benzenearsonate, the binding of the doubly charged ion apparently is also largely what is being observed because of the qualitative similarity in the effect of nitro and chloro 'The P-0 bond length in KHZPOI is 1.55"A (West, 1930). The A s - 0 bond length is 1.74"A in KH2As04 (Helmholz and Levin, 1942).
substituents on the binding of benzenephosphonateand 1)enzenearsonateby anti-R, antibody. Any contribution of antibodies directed against the singly charged hapten is difficult to define. Although the relative binding constants of the benzenearsonates are larger than the corresponding constants of the benzenephosphonates and this might be because of interaction of the singly charged arsonate with the corresponding antibody, it might also be owing to the difference in size of the arsonate and phosphonate.6 The reason why we appear to be studying anti-R, antibody that is directed against the doubly charged ion may be that, even though a large proportion of the arsonate hapten in the injected antigen a t physiological p H is in the singly ionized form, the proportion of antibody formed in response to the singly charged ion present in the pool of serum used is low.' Another factor that may be involved is that the total antigen concentrations for optimum precipitation of antibody formed in response to the singly charged and to the doubly charged forms are quite different, and the amount of antigen used was that which brought down the antibody directed against the doubly charged hapten. Comparison of Combining Sites of Anti-P, and AntiR, Antibodies.-There is no particular difficulty in understanding the combination of the smaller phosphonate ion with the anti-R, combining site, but how antibody formed in response to the smaller phosphonate ion can accommodate the larger arsonate ion needs explanation. This cross reaction may be caused by a greater state of hydration of the phosphonate ion, enough to compensate for difference in size of the unhydrated ions. Since phosphonate ion is smaller and has a greater charge density, it might be more strongly hydrated. In addition, some of the difference between the specificities observed for anti-P, and antiR, antibodies may also be due to differences in the energies of hydration of benzenephosphonate and benzenearsonate ions or in the configurations of the hydrated ions. In view of the fact that combining sites of either anti-R, or anti-P, antibodies (directed against the doubly charged ions) can accommodate either arsonate or phosphonate haptens equally well, the marked difference in specificities between the combining sites of anti-R, and anti-P, antibodies was unexpected. The difference in specificity may be of fundamental significance in concepts of antibody site structure. It may be that the amino acid sequence of the primary peptide chain can fold only in certain ways, and the only possible folding around the phosphonate ion yields a configuration which can accommodate the larger arsonate as well as the smaller phosphonate.
ACKNOWLEDGMENTS We wish to thank Dr. G . 0. Doak and Dr. R. K. Ingham for several of the benzenephosphonic acids which they supplied, and Mr. K. Mellenger for technical assistance.
REFERENCES Erlenmeyer, H., and Berger, E. (1932), Biochern. 2. 255, 429. Interactions of doubly charged arsonate with antibody formed in response to singly charged hapten or singly charged arsonate with antibody formed in response to doubly charged hapten were probably negligible. This situation could arise if the relative amount of antibody directed against the doubly charged ion were very high in the serum of the two rabbits that contributed the largest proportion to the pool of anti-R, serum.
Vol. 3, No. 2, Feb., 1964
POLYPEPTIDE CHAINS O F ?-GLOBULIN
Helmholz, L., and Levine, R. (1942), J. A m . Chem. Soc. 64, 354. Jaffe, H. H., Freedman, L. D., and Doak, G. 0. (19531, J. A m . Chem. Soc. 75, 2209. Kreiter, V. P., and Pressman, D. (1963), Biochemistry 2, 97. Landsteiner, K. (1945), The Specificity of Serological Reactions, revised ed., Cambridge, Mass., Harvard University Press. Lowry, 0. H., Rosebrough, N . J., Farr, A. L., and Randall, R. J. (1951), J . Biol. Chem. 193, 265. Nisonoff, A., and Pressman, D . (1958), J. Immunol. 80, 417.
279
Pauling, L., and Pressman, D. (1945), J. A m . Chem. Soc. 67, 1003. Pauling, L., Pressman, D., and Grossberg, A. L. (1944), J . Am. Chem. SOC.66, 784. Pressman, D., and Brown, D. H. (1943), J. A m . Chem. SOC. 65, 540. Pressman, D., Pardee, A. B., and Pauling, L. (1945), J. Am. Chem. SOC.67, 1602. Pressman, D., and Siegel, M. (1953), J. A m . Chem. SOC. 75, 686. Pressman, D., Single, S. M., Grossberg, A. L., and Pauling, L. (1944), J. Am. Chem. SOC.66, 1731. West, J. (1930), 2. Krist. 74, 306.
The Polypeptide Chains of Rabbit r-Globulin and Its Papain-cleaved Fragments* ERICMARLER, CHARLES A. NELSON, AND CHARLES TANFORD
From the Department of Biochemistry, Duke University Medical Center, Durham, N . C. Received September 19, 1963
It is shown by sedimentation equilibrium measurements that rabbit y-globulin consists of four polypeptide chains, two with a molecular weight of close to 50,000, and two with a molecular weight of close to 25,000. Similar measurements show that each of the three fragments of y-globulin, which are obtained by the action of papain, has a molecular weight of close to 50,000. Each fragment consists of two polypeptide chains of molecular weight close to 25,000 apiece. From these data it can be concluded that the action of papaln on the parent molecule has been to split the two heavier polypeptide chains into approximately equal halves. All of the foregoing data confirm the model of the gross structure of y-globulin which has been proposed by Porter (1962). It has recently been suggested by Porter (1962) that rabbit y-globulin probably consists of four polypeptide chains, two A chains having a molecular weight of about 50,000 each, and two B chains having a molecular weight of about 25,000 each. A similar structure has been suggested by Edelman and Benacerraf (1962) as one of several possibilities for human y-globulin. The work presented in this paper was initiated before Porter’s and Edelman’s results were available. It set out to determine the number of polypeptide chains in rabbit y-globulin, and their molecular weights, by a method which would not require separation of the chains. The result is an unequivocal demonstration of the correctness of Porter’s conclusions. In addition to determining the nature of the polypeptide chains of rabbit y-globulin itself, we have made similar studies on the fragments of the molecule which are obtained by the action of papain. Each molecule yields three fragments by this procedure: fragment 111, which is crystalline and plays no part in conferring specificity on those y-globulin molecules which are specific antibodies, and two fragments which are the carriers of antibody specificity. These latter fragments are designated fragments I or 11, depending on their ease of elution from a carboxymethyl-cellulose column at p H 5.5. We have studied fragments of type I only, but there is every reason to believe that the gross structure of fragments of type I1 is the same as that of fragments of type I (Palmer et al., 1962). *This work has been supported by a research grant (AM-04576) from the National Institute of Arthritis and Metabolic Diseases, U. S. Public Health Service, and by a research grant (G-17477) from the National Science Foundation. The authors are supported, respectively, by a Public Health Service training grant (2A-5074), by a Public Health Service post-doctoral fellowship, and by a Public Health Service research career program award.
MATERIALS AND METHODS Rabbit y-globulin has been prepared in this laboratory by a variety of methods. Physical and chemical studies which will be published at a later date indicate that most of these methods yield proteins which do not differ appreciably from each other, except that they possess somewhat different titration curves. The sedimentation velocity ciata reported in this paper were obtained from several such different preparations. The quantitative measurement of the molecular weights of the polypeptide chains of y-globulin (Table 11), which constitutes the most important result reported, was carried out on a sample of rabbit y-globulin prepared by a modification of the procedure of Gorodskaya (19501, using the reagent Rivanol (2-ethoxy-6,9diamino-acridine lactate) for the initial precipitation of the acidic proteins of rabbit serum. Excess Rivanol was removed by precipitation with approximately 1 M KI, and the crude globulin was then purified by chromatography on DEAE-cellulose (0.05 M potassium phosphate buffer,p H 7.0))precipitation with (NH4)2S04, and a second column chromatography, this time on DEAE-Sephadex. The final product was dialyzed against 0.1 M KC1 and stored as a lyophilized powder. This protein proved virtually homogeneous by sedimentation velocity, including little or none of the heavy component ( s ~ , , , ~ 10) which we have found present in most preparations of y-globulin and of specific antibodies. The fragments of y-globulin were prepared by the method of Porter (1959), except that mercaptoethanol was used as reducing agent in place of cysteine. The reaction was stopped by addition of iodoacetamide. In order to reduce the proteins to their polypeptide chains they were dissolved in 6-7 M guanidine hydrochloride and then allowed to stand a t room temperature for at least 12 hours in 0.1 M mercaptoethanol
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